Abstract Uterine leiomyomas, better known as fibroid tumors, are clinically apparent in almost 25% of women and cause major morbidity to American women with almost 200,000 surgeries performed to either remove the leiomyoma tumors or the whole uterus (hysterectomy). Ours and other groups have utilized whole exome sequencing to identify exon 2 of MED12 as a hotspot of mutations in leiomyomas. Our study on racially diverse population of American women showed that Med12 exon 2 was mutated in 100/148 leiomyomas (67%). The most common human MED12 mutation among leiomyomas of American women is a non-synonymous variant, c.131G>A, predicted to substitute a highly conserved glycine with aspartic amino acid (p.Gly44Asp). We generated novel mouse models that showed leiomyoma formation in the presence of Med12 c.131G>A nucleotide variant. The onset of tumor formation was earlier and the size of these tumors was larger when Med12 c.131G>A nucleotide variant was expressed on Med12 deficient uterine mesenchymal background. Conditional deficiency of Med12 in uterine mesenchyme did not lead to tumor formation. Our preliminary data is consistent with Med12 c.131G>A nucleotide variant acting via a gain of function genetic mechanism. In the current proposal we will build upon our preliminary studies to further understand molecular mechanisms behind Med12 c.131G>A nucleotide variant induced pathology, genomic imbalances and how Med12 c.131G>A nucleotide variant interacts with previously implicated pathways in leiomyoma formation, such as beta-catenin, REST and GPR10. The three aims in our proposal will test the following hypotheses: 1) Med12 c.131G>A nucleotide variant is a hormonally responsive gain of function mutation and interacts synergistically with other pathways implicated in leiomyoma formation, 2) Med12 c.131G>A nucleotide variant disrupts DNA binding and overall gene expression with disruption confined via tissue specific mechanisms to the myometrium and 3) Med12 c.131G>A nucleotide variant drives genomic instability observed in leiomyomas via a common set of recurring genomic imbalances. The focus on Med12 and its role in reproductive function is of great importance given recent human studies showing its association in uterine leiomyomas, including poorly understood leiomyosarcomas. We have successfully generated a mouse model of Med12 mutation that results in impressive leiomyomas and replicates well the human condition. We will use this model to better understand mechanisms behind the Med12 actions in leiomyoma formation.